Method for the determination of a nucleic acid using a control

ABSTRACT

The present invention is directed to a method for the determination of a target nucleic acid using a special control nucleic acid, a method for the amplification of a partial sequence of said target nucleic acid using primers, a special control and a kit containing said control. The sequence of these control nucleic acids are at least in part parallel-complementary to the sequence of the target nucleic. These controls have similar properties as the target nucleic acid in hybridization and amplification methods, but can be well differentiated from the target nucleic acid by their different sequence.

[0001] A method for the determination of a nucleic acid using a controlThis invention is directed to a method for the determination of a targetnucleic acid using a special control nucleic acid, a method for theamplification of a partial sequence of said target nucleic acid usingprimer(s) and a special control nucleic acid and a kit containing saidcontrol nucleic acid.

BACKGROUND OF THE INVENTION

[0002] The determination of nucleic acids has become an important toolin analytical chemistry, especially in health care. For example,infection diseases and genetic status can be easily determined on thebasis of the presence or the amount of a nucleic acid indicative of saiddisease or status in samples received from the individual. For thisreason methods were established using sequence specific hybridization ofa nucleic acid, preferably an oligonucleotide, with a target nucleicacid indicative for that disease or genetic status. Sensitive techniqueslike the branched DNA-method (U.S. Pat. Nos. 5,681,702, 5,597,909,5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670,5,591,584, 5,624, 802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and5,681,697), or methods detecting rRNA target nucleic acids (EP 0 272009), which are present in high copy numbers in an organism, can be usedfor direct detection of a target nucleic acid in a sample from thatorganism. But many target nucleic acids are present in an organism insuch low concentration, that a direct detection in a sample derived fromthat organism is not possible. Such targets need to be amplified beforedetection. Suitable amplification methods are for example LCR (U.S. Pat.Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; WO 90/01069;WO 89/12696; and WO 89/09835), cycling probe technology (U.S. Pat. Nos.5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT publishedapplications WO 95/05480, WO 95/1416, and WO 95/00667), Invader TMtechnology (U.S. Pat. Nos. 5,846,717; 5,614, 402; 5,719,028; 5,541,31 1;and 5,843,669), Q-Beta replicase technology (U.S. Pat. No. 4,786,600),NASBA (U.S. Pat. No. 5,409,818; EP-0 329 822), TMA (U.S. Pat. Nos.5,399,491, 5,888,779, 5,705,365, 5,710,029), SDA (U.S. Pat. Nos. 5,455,166 and 5,130,238) and PCR (U.S. Pat. No. 4,683,202).

[0003] In order to minimize false results in nucleic aciddeterminations, authorities in several countries require the use ofcontrol nucleic acids. Especially when using amplification methods suchcontrol nucleic acids are very important, because the amplificationprocess can be strongly influenced by the reaction conditions, whichcould lead to misleading results. Sometimes inhibitory substances arecontained in a sample, which could lead to false negative results.

[0004] In general one can distinguish external and internal controls.External controls, like classical positive and negative controls arenormally used to check whether the assay runs properly or whethercontaminants are contained. An internal control for example is usefulfor recognizing inhibitory substances possibly contained in a sample orcan be used as a quantification standard in a quantitative assay. Incontrast to an external control, which normally is tested in a separatereaction chamber, an internal control is preferably incubated in thesame reaction chamber together with the analyte to be tested. Therefore,the control or the amplified product of that control has to bedistinguishable from the analyte or from the amplified product of thatanalyte. When using an amplification method an internal control nucleicacid is being co-amplified essentially under the same reactionconditions as the target nucleic acid. These conditions include reagentconcentrations, temperature, inhibitor concentration or enzymaticactivities. Frequently used sequences for controls are derived fromhousekeeping genes (see Chelly et al. (1990) Eur. J. Biochem.187:691-698; Mallet et al. (1995) J. Clin. Microbiol. 33:3201-3208), butalso non-natural sequences are being used (Besnard et al. (1995) J.Clin. Microbiol. 32:1887-1893; Gilliland et al. (1990) Proc. Natl. Acad.Sci. USA 87:2725-2729; Wang et al. (1989) Proc. Natl. Acad. Sci. USA9717-9721).

[0005] The amplified nucleic acid derived from the internal control canbe distinguished from the amplified nucleic acid derived from the targetnucleic acid for example by their different length or hybridizationcapability to a distinct probe (for reviews see: Clementi et al. (1990)PCR Methods Applic. 2:191-196; Clementi et al. (1995) Arch. Virol.140:1523-1539). In all cases the nucleotide sequence of the internalcontrol is partially or totally different from the target nucleic acidsequence. However the sequence and the length of a nucleic aciddetermine its GC-content, secondary structures and melting temperatureand, therefore, is essential for its behavior in a hybridization andamplification reaction. A different sequence of an internal control innearly all cases result in a different behavior of the control nucleicacid compared with that of the target nucleic acid. In contrast theretoan ideal internal control should mimic exactly the target nucleic acidin order to allow a proper monitoring of the reaction.

[0006] One of the most critical aspects in an amplification reaction isthe binding of the primer to the target nucleic acid. Therefore internalcontrols are being used, which have the same primer binding sites as thetarget nucleic acid (see for example Gilliland et al. (1990) Proc. Natl.Acad. Sci. USA 87:2725-2729; Wang et al. (1989) Proc. Natl. Acad. Sci.USA 9717-9721; U.S. Pat. No. 5,219,727). This could lead to acompetition in the reaction for the primers and could result in adecreased sensitivity of the assay.

[0007] Gilliland et al. (1990) Proc. Natl. Acad. Sci. USA 87:2725-2729describe internal controls which nearly have the same nucleotidesequence as the target nucleic acid, but contain a new restrictionenzyme cleavage site or the sequence of an intron region not containedin the target nucleic acid. Due to the very high homology of theinternal control with the target, both nucleic acids as well as theamplificates can cross-hybridize with each other. Dependent on thedetection method used, this can lead to wrong results especially inquantitative assays. Also, the described methods requires elaboroustechniques like restriction enzyme digestion and agarose gelelectrophoresis of the amplified products.

[0008] U.S. patent application No. US 2001/0006800 A1 describes relatedcontrol nucleic acids which comprise the internal target sequencewithout the primer regions in an inverted orientation. This applicationdoes not describe controls which comprise the complementary targetsequence in an inverted orientation nor controls comprising targetprimer sequences in an inverted orientation. Especially it is to notethat inversion of the primer sequences would lead to sequences which dohave different Tm's and GC-contents compared to the original primersequences.

[0009] It is an object of the present invention to improve the methodsfor determination of nucleic acids, especially in avoiding all or a partof the disadvantages of the known methods.

SUMMARY OF THE INVENTION

[0010] The main aspect of the invention is to provide nucleic acidswhich mimic the properties of a target nucleic acid with regard tolength, G/C content, secondary structure, and folding kinetic andfurther more, but which can be distinguished easily from the respectivetarget nucleic acid by their sequence. To achieve this, a sequence isconstructed, which covers essentially the region of said target nucleicacid to be determined or the complementary strand of said target nucleicacid region and which sequence is at least in partparallel-complementary to a strand of the target nucleic acid or to astrand complementary thereto. The parallel-complementary part or partsof the control nucleic acid sequence can extend from small stretchese.g. at least 8 nucleotides, preferred at least 10 nucleotides to thecomplete target nucleic acid region to be determined in the mostextended case. In a preferred aspect of the present invention theparallel-complementary part or parts contain probe binding sitesequence(s).

[0011] In another preferred aspect, particularly, if the target nucleicacid region to be determined is amplified for determination, the controlnucleic acid according to the present invention could also containprimer binding site(s) being parallel complementary to the respectiveprimer binding site(s) of the target nucleic acid region.

[0012] Such control nucleic acids can be used for example as internalcontrols in hybridization and amplification methods and are thereforeuseful in the chemical analytic and medical diagnostic field.

[0013] The invention is also related to a method for the amplificationof a target nucleic acid region in a sample comprising the step:

[0014] amplifying said target nucleic acid region and a known amount ofcontrol nucleic acid, said control nucleic acid covering essentiallysaid target nucleic acid region to be amplified or the complement ofsaid target nucleic acid region, whereby the region of said controlnucleic acid covering essentially the region of said target nucleic acidto be amplified or the complement of said target nucleic acid containsat least one contiguous sequence of at least 8 nucleotides beingessentially parallel-complementary to said target nucleic acid or to thecomplementary strand of said target nucleic acid.

[0015] With regard to amplification methods such control nucleic acidsbear additional advantages. If a primer-binding site of these nucleicacids is parallel-complementary to the primer binding site of thetarget, these controls mimic the properties of the primer binding to thetarget, although the sequences of primer binding sites and primers ofthe control nucleic acid are different from that of the target nucleicacid. A competition of the primers for amplification of the target andthe control is avoided and the sensitivity and linear range of the assaycan be improved.

[0016] A control as mentioned can be used in any hybridization andamplification method for determination of nucleic acids. The use of thiscontrol nucleic acid is triggered by the need for a control thatreflects the characteristics of the target nucleic acid to bedetermined, but which can be still separately detectable.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows an example of a nucleotide acid sequence and itsparallel-complementary sequence as well as their capabilities to formsimilar secondary structures. (QS: control nucleic acid to be used in aquantitative or qualitative assay)

[0018]FIG. 2 shows the prediction of secondary structures done withMfold version 3.0 (copyright 1996 Dr. M. Zuker, M. Zuker, D. H. Mathews& D. H. Turner, Algorithms and Thermodynamics for RNA SecondaryStructure Prediction: A Practical Guide, In RNA Biochemistry andBiotechnology, J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series,Kluwer Academic Publishers, (1999)) for the probes ST650 (HCV-specificprobe), ST650pc (parallel complementary to ST650) and ST2535 (unrelatedsequence).

[0019]FIG. 3 shows the prediction of secondary structures done withMfold version 3.0 for the primers ST280 (HCV-specific), ST280pc(parallel complementary to ST280), ST778 (HCV-specific) and ST778pc(parallel complementary to ST778).

[0020]FIG. 4 shows the prediction of secondary structures done withMfold version 3.0 for the HCV-amplicon, QS(pc)HCV-amplicon (parallelcomplementary to HCV-amplicon) and QSHCV-amplicon (control ampliconcontaining a scrambled nucleotide sequence, a ST2535 probe bindingsequence, a ST280 and ST778 primer binding site and being of the samelenght as the HCV-amplicon).

[0021]FIG. 5 shows the annealing curves of the amplified nucleic acidderived from the different internal standards QSHCV-amplificate (Seq. IDNo. 14) and QS(pc)HCV amplificate (Seq. ID No. 13) compared with theHCV-1b amplificate

DETAILED DESCRIPTION OF THE INVENTION

[0022] The invention is mainly based on the observation that a firstnucleic acid having a sequence being parallel complementary to anothersecond nucleic acid sequence has very similar hybridization propertiesdefined by its length, GC-content, Tm and secondary structures comparedto the first nucleic acid sequence, although the sequence is completelydifferent. Such a nucleic acid can be used as control nucleic acid fordetermination of a target nucleic acid, because the control nucleic acidand the target nucleic acids and its amplificates have a very similarhybridization behavior, but are still separately detectable by theirdifferent sequences.

[0023] A preferred control nucleic acid according to the presentinvention covers essentially the region of the target nucleic acid to bedetermined characterized in that the region of said control nucleic acidcovering essentially the region of said target nucleic acid to bedetermined or the complement of said target nucleic acid contains atleast one contiguous sequence of at least 8 nucleotides beingessentially parallel-complementary to said target nucleic acid or to thecomplementary strand of said target nucleic acid.

[0024] Covering essentially the region of the target nucleic acid to bedetermined in this context means, that this region of the controlnucleic acid consists of one or more parts, whereby the sequence of eachpart is essentially identical or essentially parallel-complementary tothe according part of the target nucleic acid region to be determined orto the complementary strand. Therefore, this region of the controlnucleic acid is either essentially identical, essentiallyparallel-complementary or in part essentially parallel-complementary,whereas the other part is essentially identical to the relevant regionof the target nucleic acid region to be determined or to thecomplementary strand of the target nucleic acid. Therefore this controlnucleic acid region has essentially identical hybridization propertiescompared with the region of the target nucleic acid region to bedetermined or the complementary strand of the target nucleic acid.Slight changes in secondary structure behavior can occur if too manyessentially parallel-complementary and essentially identical parts arecombined in the control nucleic acid, whereas this does not effect theGC-content. In order to guarantee that the behavior of the control stillmimics the behavior of the target, it is preferred that the controlnucleic acid region covering the region of the target nucleic acid to bedetermined consists of fewer than 10, more preferred fewer than 6 partsbeing either essentially parallel-complementary or essentiallyidentical.

[0025] Since essentially identical or identical sequences cannot easilybe distinguished, it is very important that the region of the controlnucleic acid covering the region of the target nucleic acid to bedetermined contain at least one contiguous sequence of at least 8nucleotides, more preferred at least one sequence of at least 10nucleotides being essentially parallel-complementary to said targetnucleic acid sequence in order to allow a distinct determination oftarget nucleic acid and control nucleic acids or their amplificates forexample by probe hybridization.

[0026] A part of a first nucleic acid is parallel complementary to asecond nucleic acid or a part of it, if the sequence of that firstnucleic acid is identical with the sequence of the complementary strandof the second nucleic acid sequence or a part thereof when the sequenceof the complementary strand of the target nucleic acid sequence is readin reverse orientation. For natural nucleic acids this means reading thesequence from the 3′-end to the 5′-end. An example is given in FIG. 1.The parallel complementary sequence of the nucleic acid sequence5′-AGCGCATGCCAGATTACTGGC-3′ (Seq. ID No.1) is5′-TCGCGTACGGTCTAATGACCG-3′. (Seq. ID No.2)

[0027] The parallel complementary sequence to the complement strand is

[0028] 5′-CGGTCATTAGACCGTACGCGA-3′ (Seq. ID No. 17).

[0029] The parts of the control nucleic acids according to the presentinvention need not to be 100% identical or 100% parallel-complementaryto the target nucleic acid, although this case is preferred. It issufficient, if these parts are either essentially identical oressentially parallel-complementary. Essentially identical means that thehomology of this part or these parts of the control nucleic acid aremore than 80%, more preferred more than 90% compared with the relevantparts of the target nucleic acid. Essentially parallel-complementarymeans that the homology of this part or these parts of the controlnucleic acid are more than 80%, more preferred more than 90%parallel-complementary compared with the relevant parts of the targetnucleic acid.

[0030] For exact determination of homology and complementarity it ispreferred to use suitable computer programs like FastA (Pearson andLipman (Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988); WisconsinPackage(™) version of FastA, default settings: wordsize 2(p-p), 6(n-n);Don't show scores whose E( ) value exceeds 10.0(p-p), 2.0(n-n)).

[0031] Control nucleic acids according to the present invention are alsouseful in hybridization and amplification reactions for determination oftarget nucleic acids with related sequences like allelic forms. For thispurpose it is not necessary to synthesize a new control for each allelicform of a target nucleic acid.

[0032] A control nucleic acid according to the present invention to beused in a hybridization method as a pure control to monitor for exampleone probe hybridization step covers essentially the region of the targetnucleic acid to be determined characterized in that the region of saidcontrol nucleic acid covering essentially the region of said targetnucleic acid to be determined or the complement of said target nucleicacid contains at least one contiguous sequence of at least 8 nucleotidesbeing essentially parallel-complementary to said target nucleic acid orto the complementary strand of said target nucleic acid. The mostimportant region in such methods is the region of the target nucleicacid actually bound by the target-specific probe, which is actually theregion of the target nucleic acid being determined. Therefore, in mostcases it is sufficient that this region is being covered by the internalcontrol. In case this region is shorter than 15 nucleotides, morepreferred shorter than 10 nucleotides it is preferred that the relevantregion of the control nucleic acid is essentially parallel-complementaryto the probe hybridization region of the target nucleic acid. If theprobe hybridization region of the target nucleic acid is longer than 15nucleotides, more preferred longer than 10 nucleotides, the relevantregion of the control nucleic acid may be either fullyparallel-complementary to the probe hybridization region of the targetnucleic or the region be in part essentially parallel-complementary,whereas the other part(s) is essentially identical. Using an essentiallycomplementary, preferred a complementary probe for hybridization withsuch a control nucleic acid it is possible to mimic the hybridization ofthe target nucleic acid with its probe, but still allow a separatedetermination of both hybridization complexes.

[0033] An amplification control allows to monitor an amplificationprocess. When looking for example at a typical PCR-assay this caninclude primer-hybridization steps, elongation of the primers,amplification efficiency or/and also further probe-hybridization steps.A preferred amplification control according to the present invention cancontain regions flanking the region covering essentially the region ofthe target nucleic acid being amplified, because the preferred primersused for amplification of the control are either essentiallyparallel-complementary or essentially identical, more preferredparallel-complementary or identical to the primers for amplification ofthe target dependent on the sequence of the control. Therefore theamplificate of the control nucleic acid does not contain the flankingregions of the control but only the region which essentially covers theregion of the target nucleic acid being amplified and the length of bothamplificates are essentially identical.

[0034] The region of the control nucleic acid which essentially coversthe region of the target nucleic acid to be amplified can be built ofseveral parts which are either essentially parallel complementary oressentially identical to the according part of the target nucleic acidas described above. Preferred combinations of the control nucleic acidcovering the region of the target nucleic acid to be amplified arelisted in table 1: Probe-site(s) Non-probe/non- Primer-site(s) (ifpresent) primer region Id Pc Id Id Pc Pc Pc Pc Pc Pc Pc Id Pc Id Pc PcId Id

[0035] According to the invention only the sequence of the bases isimportant, whereas the backbone need not to necessarily be the naturalsugar-phosphate backbone. Therefore it is also possible to use nucleicacids as controls according to the present invention having a modifiedbackbone or a non natural backbone like Peptide Nucleic Acid (WO92/20702). When using control nucleic acids containing base analogues itshould be taken care that theses base(s) have similar properties to thecomplementary natural base contained in the relevant position of thetarget nucleic acid sequence in order to mimic the hybridizationbehavior of the target nucleic acid as closely as possible. Also itshould be taken care that when using a control nucleic acid as anamplification control nucleic acid, the nature of the control nucleicacid should allow the amplification of the control nucleic acid with theamplification method used.

[0036] A control nucleic acid according to the present invention can beconstructed on the basis of the sequence of the coding strand of RNA orDNA, or the strand complementary thereto. The controls can be forexample chemically synthesized and cloned in suitable vectors likeplasmids, phage nucleic acids, or in the genome of bacteria or virusesor be used as they are constructed. Such control nucleic acids can beamplified in vitro or be produced in bacteria transfected with thevectors containing the nucleic acid sequence of the controls. Suchcontrol nucleic acids can be used directly in the test or be packaged,for example as armored RNA (U.S. Pat. No. 5,677,124) or be packaged inliposomes.

[0037] The target nucleic acid is the nucleic acid to be determined.This can be of any origin, for example of viroid, viral, bacterial, orcellular origin. It can be derived from solutions, like blood, serum,plasma or urine, from suspensions, fixed on solids, cell containingmedia, cell smears, fixed cells, tissue sections or fixed organisms.Preferably the nucleic acid to be determined is in solution. The nucleicacid to be determined can further be a nucleic acid derived from thetarget nucleic acids, for example by recombination, fragmentation,amplification and/or cDNA formation from RNA.

[0038] The target nucleic acid is usually brought into available form byprocessing the original sample with one of various methods. Thiscomprises for example change of pH (alkaline), heating, cyclic changesof temperature (freezing/thawing), change of the physiological growingconditions, use of detergents, chaotropic salts or enzymes (for exampleproteases or lipases), alone or in combination. If the control nucleicacid according to the present invention is added to a sample prior orduring the sample preparation step, these control nucleic acids can alsobe used as controls for the sample preparation in addition to thesubsequent amplification and/or hybridization step. For this purpose thecontrol nucleic acid can be added to the sample as DNA or RNA dependingon the nature of the target nucleic acid. It can also be packagedsimilarly like the target nucleic acid, which is often contained in thesample attached to proteins or other cellular or viral components. Inorder to mimic the target nucleic acid more closely, the control nucleicacid may be packaged for example in a protein coat, as for example inarmored RNA.

[0039] The control nucleic acids according to the present invention canbe used for example as controls, preferably as internal controls inhybridization methods like the branchedDNA method or the determinationof ribosomal RNA targets or may be also useful for array hybridizationmethods.

[0040] They are also useful for example as controls, preferably asinternal controls, in target amplification methods, like TMA, SDA,NASBA, LCR, and PCR. The preferred method is PCR. In principle, a targetnucleic acid for example indicative for an infectious agent or a geneticstatus is used as a template to which a primer can sequence specificallybind under suitable reaction conditions. Dependent on the method usedthe primer can be for example extended with nucleotide monomers using apolymerase or ligated with a further primer hybridized nearby. In casesuch an amplification product itself can directly or indirectly serve asa template one can amplify the target nucleic acid exponentially. Alsoknown are linear amplification methods. The amplification products canbe detected directly, for example by agarose gel electrophoresis or byperforming a further hybridization reaction with at least one sequencespecific probe.

[0041] A primer according to the present invention is a molecule capableof being extended or modified preferably by enzymes, more preferably bya polymerase of for instance procaryotic origin, when hybridized to anucleic acid template. When using PCR thermostable polymerases, like T.aquaticus DNA-polymerase, are preferred. The extension addsmononucleotide units from monodesoxyribonucleosidetriphosphates to oneend of said primer, preferably the 3′-OH-terminal end. The overalllength and base sequence of a primer is dictated by the requiredspecificity of the amplification reaction. Preferred primer lengths forperforming PCR are from 10 to 40, most preferred from 15 to 30 basecontaining subunits, selected from mononucleotides and/or nucleic acidanalog monomers. In general primers of that length are also useful forother amplification methods. If more than one primer is used foramplification, for example when using PCR or amplifying multiple targetnucleic acids in one reaction, preferably primers are used which cannothybridize to each other, because they do not contain any stretch of morethan 5 consecutive complementary bases.

[0042] For PCR the locations of hybridization of the primers aretypically chosen such that there is a stretch of at least 10, but notmuch more than 1000, preferably from 100 to 500 nucleotides betweentheir original 3′-ends, in the hybrid of the extension products.

[0043] Generally, it is important that a primer binds sufficientlystrong to the target nucleic acid in order to allow amplification underthe reaction conditions, but care must be taken that the primerpreferably only binds to the target nucleic acid and binding to othernucleic acids which may be contained in a sample should be avoided.Therefore primers are preferred which are 10 to 40 nucleotides long,more preferred 15 to 30 nucleotides long. Preferably the primers areoligonucleotides.

[0044] A probe is a molecule used for the determination of a targetnucleic acid or an amplified target nucleic acid. It can also be used todetermine the sequence of a target nucleic acid or amplified targetnucleic acid. For this purpose probes can be used which have differentsequences. Such probes can also be used for example for thedetermination of different allelic forms of a target nucleic acid or fora genus and species-specific determination of a target. Probes arepreferably oligonucleotides, but also analogues, for example PNA, can beused. In order to allow a sequence specific hybridization the probes arepreferably longer than 10 nucleotides, even more preferred have a lengthof 10 to 40 nucleotides. In order to allow a detection of the targetnucleic acid, the amplificates and the according probe-hybrizationcomplexes, the probes or/and the primers used can be coupled to groupswhich can be detected directly or indirectly or which allow animmobilization of the target nucleic acid, the amplificates and theaccording probe-hybrization complexes to a solid phase.

[0045] A label is generally known to a man skilled in the art as being agroup which is detectable or can be made detectable for determining thepresence of an analyte. Well-known labels are fluorescent labels, likefluoresceine, electro-chemiluminescent labels, like ruthenium complexes,or moieties that can be recognized by another molecular entity, likehaptens which can be recognized by an antibody raised against thishapten or moieties that can be immobilized, like biotin (to streptavidincoated solid phases, like tubes or beads). Labels can be for examplebound to the probe(s) or primer(s), or can be incorporated into theamplified nucleic acids as labeled nucleoside-tri-phosphate units. Alsolabels can be used which allows the detection of the target nucleic acidor amplified target nucleic acid by other means for example byintercalation of that label or a labeled intercalator into doublestranded DNA. Labels can either be used for detection of the primers orprobes or any product having incorporated said primer, to determine thehybrid formed by said primer or probe with a nucleic acid to bedetermined or an amplified target nucleic acid incorporated labelednucleotides.

[0046] A solid phase is a solid not substantially soluble in thereaction mixture, for example in the form of a bead, a net, the innersurface of a tube, a microtiter plate or a chamber of a device. It isessentially used to contain the reaction mixture, but in case ofintention to bind an immobilisable probe, primer or control nucleic acidto it, it may contain on its surface reagents or a coating being capableto recognize and bind a moiety of said probe, primer or control nucleicacid.

[0047] Using heterogeneous detection methods, the determination, forexample by probe hybridization, is performed after the amplificationstep. The hybridization complex can be detected in solution or afterimmobilization on a solid phase. Also known are homogenous detectionformats, which allows to determine the amplified nucleic acid in thereaction mixture without further separating or washing steps. For thispurpose several probe hybridization formats are known, like Taqman (U.S.Pat. Nos. 5,210,015 and 5,487,972), Fluorescence Resonance EnergyTransfer (U.S. Pat. No. 4,996,143), Molecular Beacon (WO-95/13399, U.S.Pat. Nos. 5,119,801 and 5,312,728), Sunrise (U.S. Pat. No. 5,866,336),Scorpions (PCT/GB98/03521). Such methods can be used to detect the levelof synthesized amplificate over the whole amplification reaction. Whenusing an internal control, for example an internal control according tothe present invention, in a reaction or for determination of multipletargets the signals measured for each target and control can bedistinguished from each other. For that reason one can use for exampledifferent labels attached to the different probes or primers, whichallows a simultaneous measurement of the different targets and controlsor the amplified nucleic acids derived from these nucleic acids in thereaction mixture.

[0048] The control nucleic acid according to the present invention is anucleic acids, that may be chemically synthesized, may be cloned,amplified or isolated by other means known in the art. It is preferablymade of RNA- or DNA-monomers, but can also contain natural ornon-natural base or sugar analogues. Due to the parallel complementarypart(s) the control nucleic acids have a different sequence comparedwith the sequence of the target nucleic acid and in most cases suchsequences have to be produced synthetically at least once by knownmethods, like the phosphoramidite technology. In order to allow theproduction of longer nucleic acids according to the present inventionone can synthesize short, about 40 to 120 bases long oligonucleotideswhich overlap both strands of the desired control nucleic acid in astaggered mode. After hybridization of these oligonucleotides and afollowing ligation step the nucleic acid can for example be cloned intoa vector or can be used directly as described herein. The cloned nucleicacid can be amplified for example in bacteria and isolated usingstandard methods (Sambrook et al., Molecular Cloning: A laboratoryManual, Cold Spring Harbour Laborator, 2001, ISBN 0879695765 ). If thenucleic acid is an RNA it can be synthesized for example by in vitrotranscription using a vector, which contains a suitable promotersequence, like the T7 phage promoter sequence. The nucleic acids may beused as naked nucleic acid or be packaged in particles, for example asarmored RNA (U.S. Pat. No. 5,677,124), or be embedded in viroid, viral,bacterial, or cellular organisms.

[0049] Such nucleic acids are for example useful for controlling nucleicacid hybridization and amplification reactions. They can be used toidentify false negative results due to inhibitors present in a sample oras standards in a quantitative assay. The control can be processed in aparallel reaction in different vessels (external control) orco-processed in the same reaction vessel (internal control) as thetarget, whereas the latter case is preferred.

[0050] The essentially parallel complementary part(s) in the controlconstruct can include one or more probe binding site or/and one or moreprimer binding site or/and one or more primer/probe flanking regions, inthe most extended case the whole sequence of the corresponding targetnucleic acid.

[0051] This means that during processing of a control nucleic acidaccording to the present invention primers or/and probes can be used,that are essentially parallel-complementary, preferredparallel-complementary to those used for processing of the targetnucleic acid. Corresponding primers and probes exhibit same GC contentand hybridizing properties, that mainly reflect probe- andprimer-hybridization efficiencies and amplification efficiencies, but nocompetition with regard to these reaction components occurs, because theprimers or/and probes binding to the control nucleic acid cannot bind tothe target nucleic acid and vice versa.

[0052] In case of only low amounts of target nucleic acid in a samplesuch a competition could eventually lead to an absence of amplificationof the target or reduced probe-binding. Therefore, the use ofparallel-complementary primers and probes can increases sensitivity andlinear range of an assay. Although it should be noted, that usingidentical primers for amplification of the target nucleic acid and thecontrol nucleic acid bears the advantage that one set of primers issufficient and a direct control of the target-specific primers ispossible.

[0053] In a qualitative test, the control nucleic acid of the presentinvention can be used to identify false negative results, preferentiallyused as an internal control nucleic acid. Due to its ability to mimicthe target very close, it's subjected to inhibitors in the same manner.Especially, when the control nucleic acid is added to the reaction inlow concentration, because in this case the system reacts very sensitiveto the occurrence of inhibitory substances. Such control nucleic acidscan also be used as standards, preferably as an internal standard, forexample as a mean for standardizing of results of parallel experiments.

[0054] In a quantitative test, the control nucleic acid of the presentinvention can be used as a quantitative standard to determine startinglevel of a target nucleic acid contained in a sample. A known amount ofthe standard nucleic acid is being co-amplified essentially under thesame reaction conditions together with a target-nucleic acid, preferredas an internal quantitative standard. After amplification or, in case ofa homogeneous detection, during the amplification reaction, the amountof amplified target nucleic acid and the amount of amplified standardnucleic acid is determined. Using those and knowing the initial amountof standard nucleic acid in the reaction it is possible to calculate theinitial amount of target nucleic acid, which was present in the sampleprior to amplification. Several methods for quantification are known inthe art, including for example concepts using internal standard curve,external standard curve and Payan-Model (see for example Haberhausen etal., Journal of Clinical Microbiology, Vol 36, p. 628 to 633). It shouldbe noted that it is not necessary to measure absolut amounts ofamplificates synthesized. For most purposes relative amounts aresufficient, which can be determined for example by hybridization of theamplificates with labeled probes and measuring the signal intensity ofthe hybridization complexes.

[0055] In a preferred embodiment, the control nucleic acids of thepresent invention are used in the polymerase chain reaction (PCR),described in U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,965,188, Saiki etal., Science 230:1350-1354; mullis et al., 1986, Cold Spring HarborSymp. Quant. Biol. 51:263-273; and Mullis and Faloona, 1987, MethodsEnzymol. 155:335-350. However the invention is not restricted to anyhybridization or amplification method.

[0056] The control nucleic acid can be added at certain stages of thereaction. Usually nucleic acids of a sample, for example blood, serum,sputum or tissue sample are at least partially purified by a samplepreparation method known in the art. The particular method used is not acritical part of the present invention. The control nucleic acid can beadded prior to sample preparation allowing the control nucleic acid tobe co-processes with the target nucleic acid contained in the sample.This allows to monitor the sample preparation step in addition to thefollowing amplification or/and probe hybridization step.

[0057] In the alternative the control nucleic acid can also be added tothe amplification reaction mixture after sample preparation in order toallow monitoring of the a co-amplification of the target nucleic acidand the control nucleic acid, but can also be added after amplification,at the probe-hybridization step, to monitor the hybridization step.

[0058] Preferably for amplification of the control nucleic acidprimer(s) are used which are either essentially identical or essentiallyparallel-complementary to the primers used for amplification of thetarget nucleic acid. As such control specific primers have essentiallythe same melting temperature as the respective target-specific primers,it is easier to find an appropriate temperature cycle profile suitablefor amplification of the target nucleic acid and the control nucleicacid compared with other multiplex amplification reactions. The sameapplies if probes are used for the determination of control nucleicacids or amplified control nucleic acids, the probes being essentiallyparallel-complementary compared with the probes used for determinationof the target nucleic acid or amplified target nucleic acids.

[0059] When amplifying target nucleic acids and control nucleic acidsusing PCR, the reaction mixture typically in addition to target nucleicacid contains the control nucleic acid, specific primers and optionallyprobes for homogenous detection and further reaction components likeappropriate buffers (for example Tricine), divalent cations like Mg²⁺ orMn²⁺, a polymerase, preferably a thermostable polymerase, and the fourdesoxyribonucleotides (dATP, dCTP, dGTP and dTTP). The amplificationreaction can be performed with a thermocycler known in the art, forexample an PE 9600 (or ABI Prism 7700) (Perkin Elmer Corp.) using atemperature cycle profile allowing denaturing double stranded nucleicacid, sequence-specific hybridization of the primers to the target andcontrol and elongation of the primers by the polymerase. Thistemperature cycle can be repeated as often as necessary to allowsynthesizing sufficient amounts of amplified nucleic acids fordetermination. Usually 30 to 40 cycles are performed.

[0060] In case the amplified nucleic acids are determined by probehybridization following amplification, the amplified nucleic acids aredenatured for example by heat or alkali, neutralized (if necessary), theprobe(s) are added and hybridization of the probe to the amplifiednucleic acids is performed under suitable conditions (temperature andchemical milieu). Such conditions can be determined easily by an expertmaking a few comparative experiments. The specific hybridizationcomplexes can be detected using labels and other detection means asdescribed herein.

[0061] In case of a homogenous determination format, the amplifiednucleic acids are detected in the amplification reaction mixturepreferably during the amplification reaction. If the amplified nucleicacids are determined using specific probes, the probes are added to theamplification reaction prior to starting the amplification reaction. Inaddition such a determination format bears the advantage, that usuallythe tube need not to be opened after amplification and contaminationrisks are minimized. The hybridization of probes to the amplifiednucleic acids during the amplification reaction can be monitored usingdifferent methods. Very often the probes are labeled with fluorescentlabels, which can be detected during the reaction if suitable detectorsare integrated into the thermocycler (i.e. an ABI Prism 7700, PerkinElmer). In order to allow the detection only of probes, which have beenhybridized or hybridize to the specific amplified nucleic acid, severalformats have been established, which are referenced herein. Using theTaqMan-format the hybridized probe is partially digested by thepolymerase cutting a first quenching label from the 5′-end of the probe,which allows to measure the signal emitted by a second, preferablyfluorescent label attached to the probe. This fluorescence signal isused as a measure for the amount of specific amplified nucleic acidsynthesized in the reaction. As the probe specific to the controlnucleic acid preferably has another fluorescent label than the probespecific to the target nucleic acid emitting light having anotherwavelength, the signals of both probes can be measured separately withinthe same reaction tube. The two signals can be used to calculate, forexample, the initial amount of target nucleic acid present in thesample.

[0062] A preferred subject of the present invention is a method forquantitation of a target nucleic acid comprising the steps

[0063] a) amplifying a region of said target nucleic acid and a knownamount of control nucleic acid, said control nucleic acid coveringessentially said target nucleic acid region to be amplified or thecomplement of said target nucleic acid region, whereby the region ofsaid control nucleic acid covering essentially said target nucleic acidregion to be amplified or the complement of said target nucleic acidcontains at least one contiguous sequence of at least 8 nucleotidesbeing essentially parallel complementary to said target nucleic acid orto the complementary strand of said target nucleic acid,

[0064] b) detecting a signal indicative for the amount of amplificationproduct obtained from said control nucleic acid and detecting a signalindicative for the amount of amplification product obtained from saidtarget nucleic acid

[0065] c) calculating the amount of said target nucleic acid using theknown amount of said control nucleic acid, the signal indicative for theamount of amplification product obtained from said control nucleic aciddetermined in step b) and the signal indicative for the amount ofamplification product obtained from said target nucleic acid determinedin step b).

[0066] A further subject of the present invention is a control nucleicacid in a reaction for the amplification of a target nucleic acidregion, said control nucleic acid covering essentially the region ofsaid target nucleic acid to be amplified or the complement of saidtarget nucleic acid region, whereby the region of said control nucleicacid covering essentially the region of said target nucleic acid to beamplified or the complement of said target nucleic acid contains atleast one contiguous sequence of at least 8 nucleotides beingessentially parallel complementary to said target nucleic acid or to thecomplementary strand of said target nucleic acid.

[0067] A further subject of the present invention is a composition ofmatter comprising a target nucleic acid and a control nucleic acid,which composition is present in a hybridization or amplificationreaction mixture for detecting the target nucleic acid region andwhereby said control nucleic acid covers essentially the region of saidtarget nucleic acid to be determined or the complement of said targetnucleic acid region characterized in that the region of said controlnucleic acid covering essentially the region of said target nucleic acidto be determined or the complement of said target nucleic acid containsat least one contiguous sequence of at least 8 nucleotides beingessentially parallel complementary to said target nucleic acid or to thecomplementary strand of said target nucleic acid.

[0068] A further subject of the present invention is a kit foramplification of a target nucleic acid comprising of an instructionmanual and at least one container containing at least a control nucleicacid, whereby said control nucleic acid covers essentially the region ofsaid target nucleic acid to be amplified or the complement of saidtarget nucleic acid region characterized in that the region of saidcontrol nucleic acid covering essentially the region of said targetnucleic acid to be amplified or the complement of said target nucleicacid contains at least one contiguous sequence of at least 8 nucleotidesbeing essentially parallel complementary to said target nucleic acid orto the complementary strand of said target nucleic acid. Furtherpreferred kits contain in addition to the manual and the control nucleicacid other reaction components like target or control specific probes,target or control specific primers, reaction buffers,nucleoside-triphosphates or enzymes like a polymerase. These componentscan be packaged into the first container of such a kit, but can also becontained in one or more further containers. Also preferred is a kituseful for detection of a target nucleic acid using hybridizationmethods which contains an instruction manual and at least one controlnucleic acid according to the present invention.

[0069] A further subject of the present invention is the use of acontrol nucleic acid as a control in a reaction for amplification of atarget nucleic acid region or in a hybridization reaction fordetermination of a target nucleic acid region, whereby said controlnucleic acid covers essentially the region of said target nucleic acidto be amplified or hybridized or the complement of said target nucleicacid region characterized in that the region of said control nucleicacid covering essentially the region of said target nucleic acid to beamplified or hybridized or the complement of said target nucleic acidcontains at least one contiguous sequence of at least 8 nucleotidesbeing essentially parallel complementary to said target nucleic acid orto the complementary strand of said target nucleic acid.

[0070] The present invention is exemplified by the following examples:

EXAMPLES Example 1

[0071] Design of a Probe Sequence, that is Parallel Complementary to theHCV Target Probe Sequence

[0072] For detection of HCV nucleic acid in a Taqman assay the followingprobe, binding to the HCV-amplicon (Seq. ID No. 10) can be used: ST6505′ (Cy5-) CGG TGT ACT CAC CG(FAMs) TTC CGC AGA CCA CTA TGG C-PO₄-3′:(Seq. ID No 3)

[0073] Cy5: Oligonucleotid-derivatisation withPentamethin-di-indocarbocyanin using Alkylphosphatidyl-Linker (PharmaciaBiotech Cy5-N-ethyl-Phosphoramidit)Fams: Oligonucleotid-derivatisationwith 6-Carboxy-fluorescein using2-(Amino-cyclohexyl-)propan-1,3-diol-Linker (BiogenexCX-FAM-Phosphoramidit)

[0074] All HCV specific sequences enclosed in this application arederived from the sequence of the HCV type I genome (NCBI, GenbankAccession No. AF054249 or AJ000009).

[0075] At best the probe for internal control detection mimics thetarget probe exactly, but is distinguishable of the target probe. Theparallel complementary sequence of the target probe, called ST650pc(Seq. ID No. 5), can be used as such an internal control probe. ST650pchas the same GC-content, GC/AT sequence, length, secondary structure andtherefor the same melting temperature as the target probe. Furthermore,since G and A are substituted by C and T, respectively, and vice versa,it is distinguishable to the target probe. ST2535 (Seq. ID No. 4)contains a sequence which is not related to the sequence of HCV, e.g. isnot essentially identical or essentially parallel-complementary to thesequence of HCV. An internal control containing a binding sequence forST2535 can be used together with the probe ST2535 to control the targetnucleic acid determination reaction. Probes and controls containing asequence unrelated to that of the target nucleic acid sequence are oftenused in the prior art. In contrast thereto ST650pc (Seq. ID No. 5) hasthe potential to mimic target probe perfectly. ST650pc (Seq. ID No. 5)probe can be used with all internal control or quantification standardconstructs containing a ST650pc binding sequence. ST2535 5′ (Cy5-) TGGACT CAG TCC T(HEXs)T GGT CAT CTC ACC TTC T-PO4 3′: (Seq. ID No.4)

[0076] HEXs: Oligonucleotid-Derivatisation withHexachloro-6-carboxy-fluorescein using 2-(Amino-cyclohexyl)propan-1,3-diol-Linker (Biogenex CX-HEX-Phosphoramidit) ST650pc5′ (CY5-) GCC ACA TGA GTG GC(HEXs) AAG GCG TCT GGT GAT ACC G-PO4 3′:(Seq. ID No.5)

[0077] Prediction of secondary structures was done using the softwareMfold version 3.0 (copyright 1996 Dr. M. Zuker, M. Zuker, D. H. Mathews& D. H. Turner, Algorithms and Thermodynamics for RNA SecondaryStructure Prediction: A Practical Guide, In RNA Biochemistry andBiotechnology, J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series,Kluwer Academic Publishers, (1999)). As can be seen in FIG. 2 thepredicted secondary structures are identical between target probe andparallel complementary probe.

Example 2

[0078] Design of Primer Sequences, that are Parallel Complementary tothe HCV Target Primer Sequences and Comparison of Likely FormedSecondary Structures Formed by Target Primers and Parallel ComplementaryPrimers:

[0079] For amplification of HCV nucleic acid the following primers areused: ST280 5′ GCA GAA AGC GTC TAG CCA TGG CGT TA (Seq ID No 6) 3′:ST778 5′ GCA AGC ACC CTA TCA GGC AGT ACC (Seq ID No 7) ACA A 3′:

[0080] The parallel-complementary sequences of these amplificationprimers are ST280 pc 5′ CGT CTT TCG CAG ATC GGT ACC TCA AT (Seq ID No 8)3′: ST778 pc 5′ CGT TCG TGG GAT AGT CCG TCA TGG (Seq ID No 9) TGT T 3′:

[0081] These designed internal primers are used for amplification of theinternal control. Amplification of the internal control isnon-competitive, since target and internal control use different primersfor amplification. Nevertheless, the designed parallel complementaryprimers are structural very close to target primers, with regard to GCcontent, GC/AT sequence, length, secondary structure and thereformelting temperature. ST280pc and ST778pc can be used as amplificationprimers for all internal control and quantification standard constructsthat are non competitive and contain appropriate primer binding sites.

[0082] Predictions of secondary structures, done with Mfold version 3.0,are identical between target primers and parallel-complementary primers(see FIG. 3).

Example 3

[0083] Construction of an Internal Control for Non-CompetitiveQualitative and Quantitative Nucleic Testing of HCV, that is ParallelComplementary to HCV Sequence:

[0084] A double stranded DNA fragment was cloned, using overlappingoligonucleotides with a length of 60-120 b, that is parallelcomplementary to a 5′-fragment of HCV type I genome including the highlyconserved 5′-UTR region. The synthesized and cloned fragment is 943 bplong and cloned into the BglII/HindIII site of an aRNA expression vector(AMBION, Austin, USA). Expression results in an armored RNA particlethat contains RNA including the gene for MS-2 coat protein, MS-2packaging signal and the parallel complementary sequence of 5′ HCVfragment. This RNA is distinguishable from the target, but due toparallel complementarity, GC-content and sequence of G or C and A or Tnucleotides are identical to the corresponding HCV fragment, which aremainly influencing secondary structure. This construct is coamplifiedand detected in the same reaction together with HCV target (Seq. ID No10) in a non competitive manner using the primers and the probementioned in example 2 and 1, respectively.

[0085] Sequence of the cloned BglII/HindIII fragment of QS(pc)HCV (Seq.ID No 11):

[0086] SEQ New: 943 bp;

[0087] Composition 203 A; 287 C; 282 G; 171 T; 0 OTHER

[0088] Percentage: 22% A; 30% C; 30% G; 18% T; 0%OTHER

[0089] Molecular Weight(kDa): ssDNA: 291.24 dsDNA: 581.5

[0090] ORIGIN BglII 1  AGATCTCCGC TGTGAGGTGG TATCTAGTGA GGGGACACTCCTTGATGACA GAAGTGCGTC 61  TTTCGCAGAT CGGTACCGCA ATCATACTCA CAGCACGTCGGAGGTCCTGG GGGGGAGGGC 121  CCTCTCGGTA TCACCAGACG CCTTGGCCAC TCATGTGGCCTTAACGGTCC TGCTGGCCCA 181  GGAAAGAACC TAGTTGGGCG AGTTACGGAC CTCTAAACCCGCACGGGGGC GCTCTGACGA 241  TCGGCTCATC ACAACCCAGC GCTTTCCGGA ACACCATGACGGACTATCCC ACGAACGCTC 301  ACGGGGCCCT CCAGAGCATC TGGCACGTGG TACTCGTGCTTAGGATTTGG AGTTTCTTTT 361  TGGTTTGCAT TGTGGTTGGC GGCAGGTGTC CTGCAGTTCAAGGGCCCGCC ACCAGTCTAG 421  CAACCACCTC AAATGGACAA CGGCGCGTCC CCGGGGTCCAACCCACACGC GCGCGAGTCC 481  TTCTGAAGGC TCGCCAGCGT TGGAGCACCT TCCGCTGTTGGATAGGGGTT CCGAGCGGCT 541  GGGCTCCCGT CCCGGACCCG AGTCGGGCCC ATGGGAACCGGGGAGATACC GTTACTCCCG 601  TACCCCACCC GTCCTACCGA GGACAGTGGG GCACCAAGAGCCGGATCAAC CCCGGGGAGT 661  CTGGGGGCCG CATCCAGCGC ATTAAACCCA TTCCAGTAGCTATGGGAATG TACGCCGAAG 721  CGGCTGGAGT ACCCCATGTA AGGCGAGCAG CCGCGGGGAGATCCCCCGCG GCGGTCCCGG 781  GACCGCGTAC CGCAGGCCCA AGACCTCCTG CCGCACTTGATACGTTGTCC CTTAAACGGG 841  CCAACGAGAA AGAGATAGAA GGAGAACCCA AACGACAGAACAAACTGGTA GGGTCGAAGG 901  CGAATACTTC ACGCGTAAAC ATGAGGATTACCCATGTAAG CTT                HindIII

[0091] Printed in bold: Amplicon (Seq. ID No 12) when using the PrimersST280pc and ST778pc for amplification.

Example 4

[0092] Comparison of Likely Formed Secondary Structures Formed by SingleStranded HCV Target Amplicon vs. Single Stranded Internal ControlAmplicon and vs. a Scrambled Internal Control Amplicon QSHCV (Seq ID No13).

[0093] The scrambled control nucleic acid (Seq ID No 13) has a ST280 andST778 primer binding site, a ST2535 probe binding site and is of thesame length as the HCV target amplicon and has the same GC-content.Folding was performed with Mfold version 3.0, see FIG. 4.

[0094] HCV- and the parallel complementary QS(pc)HCV amplicon have thepotential to form similar secondary structures. This is different fromthe structure that is formed by the scrambled internal control (QSHCV)amplicon (Seq. ID No 13) that is state of the art. Since QS(pc)HCVamplicon (Seq. ID No 12) is a very close mimic of the HCV targetamplicon, QS(pc)HCV (Seq. ID No 11) is the better choice to controltarget amplification.

Example 5

[0095] Cloning of an Internal Control for Competitive, Qualitative andQuantitative Nucleic Testing of HCV with Native Primer Binding Sites(ICSJ620HCV)

[0096] According to example 3 a synthetic 375 bpDNA fragment was clonedthat is, besides the primer binding site, parallel complementary to HCVtype I 5′UTR. The primer binding sites are identical to the targetsequence to allow a competitive PCR between target and control. Thesequence between both primers is parallel complementary in order tomimic the target. Since GC content, length and sequence of G/C and A/Tbetween the primers is identical to the target, this construct is veryclose to the target amplicon. This construct can be coamplified anddetected in the same reaction together with HCV target in a competitivemanner using target amplification primers and the parallel complementaryprobe mentioned in example 1.

[0097] ICSJ620HCV (Seq. ID No 14)

[0098] SEQ New: 375 bp;

[0099] Composition 76 A; 104 C; 117 G; 78 T; 0 OTHER

[0100] Percentage: 20% A; 28% C; 31% G; 21% T; 0%OTHER

[0101] Molecular Weight (kDa): ssDNA: 116.05 dsDNA: 231.2

[0102] ORIGIN BglII 1 AGATCTCGGT CGGGGGACTA CCCCCGCTGT GAGGTGGTACTTAGTGAGGG GACACTCCTT 61 GATGACAGAA GTGGCAGAAA GCGTCTAGCC ATGGCGTTACATACTCACAG CACGTCGGAG 121 GTCCTGGGGG GGAGGGCCCT CTCGGTATCA CCAGACGCCTTGGCCACTCA TGTGGCCTTA 181 ACGGTCCTGC TGGCCCAGGA AAGAACCTAG TTTGGGCGAGTTACGGACCT CTAAACCCGC 241 ACGGGGGCGC TCTGACGATC GGCTCATCAC AACCCAGCGCTTTCCGGTTG TGGTACTGCC 301 TGATAGGGTG CTTGCCTCGA GGGGCCCTCC AGAGCATCTGGCACGTGGAA ACATGAGGAT 361 TACCCATGTA AGCTT   HindIII

[0103] Printed in bold: Amplicon (Seq. ID No 15) when using the primersST280 and ST778 for amplification.

Example 6

[0104] Comparison of Annealing Curves Derived from HCV1b Wildtype, QSHCVand QS(pc)HCV

[0105] Related fragments of HCV-1b wildtype, QSHCV and QS(pc)HCV wereamplified and subsequently amplification products were compared in anannealing experiments using SybrGreen as intercalating agent to getinsight into folding kinetics of the amplicon 1000 IU HCV-1b RNA, QSHCVor QS(pc)HCV in comparable concentrations (IU: International Unit,WHO-Standard) 5 % DMSO 5,64 % Glycerol 50 mM Tricine (pH=8,3) 100 mMKOAc (pH=7,5) 300 μM dNTP(A,C,G) 50 μM dTTP 500 μM dUTP 20 pmolNTQ21-46-A (Aptamer, Sequence: CGA TCA TCT CAG AAC ATT CTT AGC GTT TTGTTC TTG TGT ATG ATC G-PO₄) 40 U ZO5 Polymerase 10 U UNG(Uracil-N-Glycosylase) 3 mM Mn(Ac)₂ 12 μl Sybr-Green (1:300 dilution,from Molecular Probes, Leiden Netherlands; 10000× concentrated in DMSO)ad 100 μl water DEPC-treated Primers

[0106] Primers in amplification of HCV-1b and QSHC 15 pmol ST280 40 pmolST778 Primers in amplification of QS(pc)HCV 15 pmol ST280pc 40 pmolST778pc

[0107] The polymerase ZO5 is described in U.S. Pat. Nos. 5,455,170 and5,674,738. Amplification cycles: (50° C. 240 s 59° C. 1800 s) 1x (95° C.15 s 58° C. 25 s) 5x (91° C. 15 s 58° C. 25 s) 55x Annealing curve 0.5°C. steps beginning at 90° C. cooling to 70° C. 20 s each step

[0108] Amplification and annealing curves (detected by Sybr-Greenintercalation) were done on an ABI Prism 7700 (Perkin Elmer Corp.)

[0109] Comparison of annealing curves (see FIG. 5)

[0110] Reference in the assay is the annealing curve of the HCV-1bamplicon. The internal control construct that is used in the art QSHCVshows an annealing curve with a higher melting temperature, whereas theannealing curve of the parallel complementary control sequence QS(pc)HCVis matching the annealing curve of HCV-1b amplicon nearly perfectly.

1 17 1 21 DNA Artificial Sequence Description of Artificial SequenceArtificial sequence to exemplify principle 1 agcgcatgcc agattactgg c 212 21 DNA Artificial Sequence Description of Artificial SequenceArtificial sequence to exemplify principle 2 tcgcgtacgg tctaatgacc g 213 33 DNA Artificial Sequence Description of Artificial Sequence ST650HCV specific probe sequence 3 cggtgtactc accgttccg cagaccacta tggc 33 430 DNA Artificial Sequence Description of Artificial Sequence SequenceST2535 probe sequence 4 tggactcagt ccttggtca tctcaccttc t 30 5 33 DNAArtificial Sequence Description of Artificial Sequence ST650pc probeseqeunce (parallel-complementary to ST650) 5 gccacatgag tggcaaggcgtctggtgat accg 33 6 26 DNA Artificial Sequence Description ofArtificial Sequence sequence ST280 HCV-specific primer sequence 6gcagaaagcg tctagccatg gcgtta 26 7 28 DNA Artificial Sequence Descriptionof Artificial Sequence ST778 HCV-specific primer sequence 7 gcaagcaccctatcaggcag taccacaa 28 8 26 DNA Artificial Sequence Description ofArtificial Sequence ST280pc primer parallel complement to ST280 8cgtctttcgc agatcggtac ctcaat 26 9 28 DNA Artificial Sequence Descriptionof Artificial Sequence ST778pc primer parallel-complement to ST778 9cgttcgtggg atagtccgtc atggtgtt 28 10 241 DNA Artificial SequenceDescription of Artificial Sequence DNA sequence derived by amplificationof HCV type 1 using primers ST280 and ST778 10 gcagaaagcg tctagccatggcgttagtat gagtgtcgtg cagcctccag gaccccccct 60 cccgggagag ccatagtggtctgcggaacc ggtgagtaca ccggaattgc caggacgacc 120 gggtcctttc ttggatcaacccgctcaatg cctggagatt tgggcgtgcc cccgcgagac 180 tgctagccga gtagtgttgggtcgcgaaag gccttgtggt actgcctgat agggtgcttg 240 c 241 11 943 DNAArtificial Sequence Description of Artificial Sequence QS (pc) HCV beingparallel-complement to according region of HCV type 1 genome 11agatctccgc tgtgaggtgg tatctagtga ggggacactc cttgatgaca gaagtgcgtc 60tttcgcagat cggtaccgca atcatactca cagcacgtcg gaggtcctgg gggggagggc 120cctctcggta tcaccagacg ccttggccac tcatgtggcc ttaacggtcc tgctggccca 180ggaaagaacc tagttgggcg agttacggac ctctaaaccc gcacgggggc gctctgacga 240tcggctcatc acaacccagc gctttccgga acaccatgac ggactatccc acgaacgctc 300acggggccct ccagagcatc tggcacgtgg tactcgtgct taggatttgg agtttctttt 360tggtttgcat tgtggttggc ggcaggtgtc ctgcagttca agggcccgcc accagtctag 420caaccacctc aaatggacaa cggcgcgtcc ccggggtcca acccacacgc gcgcgagtcc 480ttctgaaggc tcgccagcgt tggagcacct tccgctgttg gataggggtt ccgagcggct 540gggctcccgt cccggacccg agtcgggccc atgggaaccg gggagatacc gttactcccg 600taccccaccc gtcctaccga ggacagtggg gcaccaagag ccggatcaac cccggggagt 660ctgggggccg catccagcgc attaaaccca ttccagtagc tatgggaatg tacgccgaag 720cggctggagt accccatgta aggcgagcag ccgcggggag atcccccgcg gcggtcccgg 780gaccgcgtac cgcaggccca agacctcctg ccgcacttga tacgttgtcc cttaaacggg 840ccaacgagaa agagatagaa ggagaaccca aacgacagaa caaactggta gggtcgaagg 900cgaatacttc acgcgtaaac atgaggatta cccatgtaag ctt 943 12 241 DNAArtificial Sequence Description of Artificial Sequence Amplicon derivedfrom QS (pc)HCV using the primers ST280pc and ST778pc 12 cgtctttcgcagatcggtac cgcaatcata ctcacagcac gtcggaggtc ctggggggga 60 gggccctctcggtatcacca gacgccttgg ccactcatgt ggccttaacg gtcctgctgg 120 cccaggaaagaacctagttg ggcgagttac ggacctctaa acccgcacgg gggcgctctg 180 acgatcggctcatcacaacc cagcgctttc cggaacacca tgacggacta tcccacgaac 240 g 241 13 241DNA Artificial Sequence Description of artificial sequence Ampliconsequence derived from QS HCV (HCV amplification control having bindingsites for ST280, ST778, and ST2535) using primers ST280 and ST778 13gcagaaagcg tctagccatg gcgttagtat agtggcgtga gagcagccct tgcctcgccc 60accgcgcgtc tagaaggtga gatgaccaga ggactgagtc caatgcatgc tggctccgag 120atgctccgca aacttgccgt caacgtgact gcgtacggcg ggcgtgcccg cctggctgtg 180tatgagctgg tgaccgtgat ctggctggag gccttgtggt actgcctgat agggtgcttg 240 c241 14 375 DNA Artificial Sequence Description of Artificial SequenceICSJ620 HCV (HCV specific amplification control having a binding sitefor ST280 and ST778 and an internal region being parallel-complement toHCV) 14 agatctcggt cgggggacta cccccgctgt gaggtggtac ttagtgaggggacactcctt 60 gatgacagaa gtggcagaaa gcgtctagcc atggcgttac atactcacagcacgtcggag 120 gtcctggggg ggagggccct ctcggtatca ccagacgcct tggccactcatgtggcctta 180 acggtcctgc tggcccagga aagaacctag tttgggcgag ttacggacctctaaacccgc 240 acgggggcgc tctgacgatc ggctcatcac aacccagcgc tttccggttgtggtactgcc 300 tgatagggtg cttgcctcga ggggccctcc agagcatctg gcacgtggaaacatgaggat 360 tacccatgta agctt 375 15 242 DNA Artificial SequenceDescription of artificial sequence Amplicon derived from ICSJ620 HCV(HCV-specific amplification control) using ST280 and ST778 as primers 15gcagaaagcg tctagccatg gcgttacata ctcacagcac gtcggaggtc ctggggggga 60gggccctctc ggtatcacca gacgccttgg ccactcatgt ggccttaacg gtcctgctgg 120cccaggaaag aacctagttt gggcgagtta cggacctcta aacccgcacg ggggcgctct 180gacgatcggc tcatcacaac ccagcgcttt ccggttgtgg tactgcctga tagggtgctt 240 gc242 16 46 DNA Artificial Sequence Description of Artificial SequenceNTQ21-46-A aptamer sequence 16 cgatcatctc agaacattct tagcgttttgttcttgtgta tgatcg 46 17 21 DNA Artificial Sequence Description ofArtificial Sequence Sequence to exemplify principle 17 cggtcattagaccgtacgcg a 21

1. A method for the amplification of a target nucleic acid region in asample comprising the step of amplifying a known amount of a controlnucleic acid and said target nucleic acid, wherein said control nucleicacid comprises at least one contiguous sequence of at least 8nucleotides in length essentially parallel complementary to said targetnucleic acid region or to the complementary strand of said targetnucleic acid region.
 2. A method for quantitation of a target nucleicacid region comprising the steps of: a) amplifying said target nucleicacid region and a known amount of a control nucleic acid, wherein saidcontrol nucleic acid comprises at least one contiguous sequence of atleast 8 nucleotides in length essentially parallel complementary to saidtarget nucleic acid region or to the complementary strand of said targetnucleic acid region, b) detecting a signal indicative for the amount ofamplification product obtained from said control nucleic acid anddetecting a signal indicative for the amount of amplification productobtained from said target nucleic acid, c) calculating the amount ofsaid target nucleic acid using the known amount of said control nucleicacid, the signal indicative for the amount of amplification productobtained from said control nucleic acid detected in step b) and thesignal indicative for the amount of amplification product obtained fromsaid target nucleic acid detected in step b).
 3. The method of claim 1or 2, wherein said target nucleic acid and said control nucleic acid areamplified by PCR.
 4. The method of claim 1 or 2, wherein the amplifiedproduct is detected homogeneously.
 5. The method of claim 1 or 2,wherein said target nucleic acid and said control nucleic acid areamplified in the same reaction vessel.
 6. A control nucleic acid for usein a reaction for the amplification of a target nucleic acid region,wherein said control nucleic acid comprises at least one contiguoussequence of at least 8 nucleotides in length essentially parallelcomplementary to said target nucleic acid region or to the complementarystrand of said target nucleic acid region.
 7. The control nucleic acidof claim 6, wherein said target nucleic acid region comprises a primerbinding site and said control nucleic acid comprises a sequence that isparallel complementary to the primer binding site of said target nucleicacid or to the complementary strand of said target nucleic acid.
 8. Thecontrol nucleic acid of claim 6, wherein said target nucleic acid regioncomprises a probe binding site and said control nucleic acid comprises asequence that is parallel complementary to the probe binding site ofsaid target nucleic acid or the complementary strand of the probebinding site of said target nucleic acid.
 9. A composition comprising atarget nucleic acid and a control nucleic acid, wherein said controlnucleic acid comprises at least one contiguous sequence of at least 8nucleotides in length essentially parallel complementary to said targetnucleic acid region or to the complementary strand of said targetnucleic acid region.
 10. The composition of claim 9, wherein said targetnucleic acid comprises a primer binding site and said control nucleicacid comprises a sequence that is parallel complementary to the primerbinding site of said target nucleic acid or to the complementary strandof said target nucleic acid.
 11. The composition of claim 9, whereinsaid target nucleic acid comprises a probe binding site and said controlnucleic acid comprises a sequence that is parallel complementary to theprobe binding site of said target nucleic acid or the complementarystrand of the probe binding site of said target nucleic acid.
 12. Thecomposition of claim 9, further comprising primers for the amplificationof said target nucleic acid and primers for the amplification of saidcontrol nucleic acid.
 13. A kit for the amplification of a targetnucleic acid comprising an instruction manual, a target nucleic acid anda control nucleic acid wherein said control nucleic acid comprises atleast one contiguous sequence of at least 8 nucleotides in lengthessentially parallel complementary to said target nucleic acid region orto the complementary strand of said target nucleic acid region.
 14. Thekit of claim 13, further comprising primers for the amplification ofsaid target nucleic acid and primers for the amplification of saidcontrol nucleic acid.